1. Technical Field
The subject matter of the present invention relates to aminoalkylcarbamates of forskolin and the uses of these compounds. Specifically, the aminoalkylcarbamates may be utilized as intermediates in the synthesis of forskolin derivatives. The final derivatives or analogs may, in turn, be used in the development of in vivo and in vitro assays designed to study different proteins.
2. Background Information
Forskolin is a diterpene which can interact with a diverse group of membrane proteins including adenylate cyclase and the glucose transporter (Laurenza et al., Trends in Pharmacological Sciences 10:442 (1989)). Forskolin is a natural product and was originally isolated from methanol extracts derived from the roots of Coleus Forskohlii found on the indian subcontinent (Bhat et al., Tetrahedron Letters 19:1669 (1977)). Other diterpenes similar in structure to forskolin were isolated from the same methanol extracts including 1,9-dideoxyforskolin, 7-desacetylforskolin, 6-acetyl-7-desacetylforskolin and 9-deoxyforskolin.
Forskolin produces marked cardiotonic effects due to its ability to activate the enzyme adenylate cyclase and increase intracellular cyclic AMP (Metzger et al., Arzneim, Forsch. 31:1248 (1981)). The ability of forskolin to interact directly with adenylate cyclase is a unique property of this diterpene, and forskolin consequently has been used extensively by biomedical researchers (Seamon et al., Adv. in Cyclic Nucl. Res. 20:1-150 (1988)). The ability of forskolin to increase cyclic AMP in vivo has prompted many investigations into the therapeutic potential of forskolin to treat a number of indications including asthma, glaucoma and heart disease (Burka, Can. J. Physiol. Pharmacol. 61:681 (1983)), Caprioli et al., Invest. Opthamol. Vis. Sci. 25:268 (1984) and Briston et al., J. Clin. Invest. 74:212 (1984)).
Since forskolin is a natural product, there have been a number of investigations into the total synthesis of the diterpene. Many research groups have actively pursued different methods in developing the complete synthesis of forskolin and have utilized an Intramolecular Diels Alder construction for synthesizing key intermediates (Jenkins et al., J.C.S. Chem. Commun. p. 1423 (1984), Nicolaou et al., J.C.S. Chem. Commun. p. 421 (1984) and Ziegler et al., Tetra. Letters 25:3307 (1985)). Several groups have succeeded in the total synthesis of forskolin (Ziegler et al., J. Am. Chem. Soc. 109:8115 (1987), Hashimoto et al., J. Am. Chem. Soc. 110:3670 (1988) and Corey et al., J. Am. Chem. Soc. 110:3672 (1988)).
Currently, there is a great deal of interest in semi-synthetic analogs or derivatives of forskolin. The importance of the .alpha.-face of the molecule was defined by the inability of 1,9-dideoxyforskolin and derivatives of forskolin, with the 1- and 9-hydroxyl groups modified, to activate adenylate cyclase. (Bhat et al., J. Med. Chem. 26:486 (1983) and Seamon et al., J. Med. Chem. 26:486 (1983)).
Other derivatives of forskolin have been synthesized and tested for their ability to activate adenylate cyclase. These derivatives include ester analogs of forskolin with different acyl groups esterified at the 1.alpha.-, 6.beta.-, and 7.beta.-hydroxyl groups (Bhat et al., J. Med. Chem. 26:486 (1983)). Water soluble derivatives of forskolin have also been synthesized (Khandelwal et al., J. Med. Chem. 31:1872 (1988) and Laurenza et al., Mol. Pharmacol 33:133 (1987)). Procedures have been developed for the selective acylation of the 1-, 6-, or 7-hydroxyl groups via the specific protection of the 1-hydroxyl group with dimethylformamide acetal (Kosley et al., J. Org. Chem. 54:2972 (1989)). A method has also been developed to produce 6- and 7-carbamate derivatives of forskolin containing different groups attached to forskolin through a stable carbamate linkage (O'Malley et al., J. Org. Chem. 55:1102 (1990)). 7-Carbamate derivatives are produced by the nucleophilic attack of primary or secondary amines on a 7-acyl imidazolium intermediate of forskolin. 6-Carbamates are produced by the regioselective attack of primary and secondary amines on the 6,7-carbonate of forskolin.
Derivatives of forskolin have been synthesized and tested for their ability to activate adenylate cyclase. Initial studies demonstrated the importance of the 1- and 9-hydroxyl groups of forskolin for the activation of adenylate cyclase (Seamon et al., J. Med. Chem. 26:486 (1983)). Other derivatives of forskolin have been described that are active at adenylate cyclase (Seamon et al., J. Med. Chem. 26:486 (1983)). These include 7-acyl derivatives of forskolin that contain short alkyl chains such as 7-desacetyl-7-propionylforskolin (Seamon et al., J. Med. Chem. 26:486 (1983)). Other derivatives that can activate adenylate cyclase but are less potent than forskolin include 14,15-dihydroforskolin, 11.beta.-hydroxyforskolin, 6-acetyl-7-desacetylforskolin, and 7-desacetylforskolin (Seamon et al., J. Med. Chem. 26:486 (1983)).
Other derivatives of forskolin do not activate or are not potent at activating adenylate cyclase. 1,9-Dideoxyforskolin does not activate adenylate cyclase, and derivatives of forskolin, where the 1- and 9-hydroxyl groups are conjugated, are inactive at adenylate cyclase (Seamon et al., J. Med. Chem. 26:486 (1983)). 7-Acyl derivatives of forskolin that contain lipophilic groups are not potent at activating adenylate cyclase (Seamon et al., J. Med. Chem. 26:486 (1983)).
As mentioned above, water soluble derivatives of forskolin have been synthesized. These include 7-acyl derivatives that contain heterocyclic rings which were almost as potent at adenylate cyclase as forskolin. Water soluble derivatives of forskolin that contain heterocyclic amino acids esterified at the 6-hydroxyl group are equipotent with forskolin (Khandelwal et al., J. Med. Chem. 31:1872 (1988) and Laurenza et al., Mol. Pharmacol. 32:133 (1987)).
Derivatives of forskolin have been synthesized and used for biochemical studies. These include .alpha.-haloacetyl derivatives of forskolin such as 7-descacetyl-7-bromoacetylforskolin and 7-desacetyl-7-chloroacetylforskolin which have been used to block the forskolin binding site on adenylate cyclase (Laurenza et al., Mol. Pharmacol. 37:69 (1990)). 7-Desacetyl-7-hemisuccinylforskolin has been synthesized and coupled to solid supports (Pfeuffer et al., Proc. Natl. Acad. Sci. USA 83:3086 (1985), Pfeuffer et al., EMBO J. 4:3675 (1985) and Smigel et al., J. Biol. Chem. 201:1976 (1988)). These supports have been used for the isolation and purification of adenylate cyclase. 7-Desacetyl-7-hemisuccinylforskolin has also been used as an intermediate for the synthesis of iodinated photoactivatable derivatives of forskolin for covalently labelling adenylate cyclase (Pfeuffer et al., FEBS Lett. 248:13 (1989)).
The synthesis of forskolin analogs has been aimed predominantly at designing those derivatives of forskolin that would be active at adenylate cyclase. There is much less information available concerning the interaction of forskolin at other forskolin binding proteins. Forskolin interacts with proteins other than adenylate cyclase, and there is some information regarding the binding of forskolin and forskolin analogs at the glucose transporter. 1,9-Dideoxyforskolin and derivatives of forskolin that contain lipophilic groups esterified at the 7-hydroxyl group inhibit glucose transport in adipocyte membranes (Joost et al., Mol. Pharmacol. 33:449 (1988)). These derivatives are not active at adenylate cyclase.
7-Desacetyl-7-hemisuccinylforskolin has been used as a starting material to synthesize photoactivatable derivatives of forskolin containing radioactive iodine (Wadzinski et al., J. Biol. Chem. 262:5978 (1978)). These derivatives have been used to covalently label the glucose transporter in a variety of tissues.
Forskolin and derivatives of forskolin could be used for a number of purposes due to the ability of forskolin to interact with a number of diverse and physiologically important proteins (Laurenza et al., Trends in Pharmacol. Sci. 10:442 (1989)). However, the derivatives of forskolin that have been synthesized to date have not been designed to be specific for the different sites of action of forskolin. For example, forskolin is equipotent at activating adenylate cyclase and inhibiting glucose transport. There has not been any rationale in designing derivatives of forskolin that would be potent at adenylate cyclase and not potent at the glucose transporter.
Many of the derivatives of forskolin that have been developed have been ester analogs. These include the .alpha.-haloacetyl analogs of forskolin, 7-bromoacetyl-7-desacetylforskolin and 7-chloracetyl-7-desacetylforskolin and derivatives utilizing 7-desacetyl-7-hemisuccinylforskoklin as an intermediate. There are potential problems in using ester-analogs for in vivo and in vitro studies. For example, ester analogs of forskolin are susceptible to hydrolysis and rearrangement under mildly basic conditions (Bhat et al., J. Chem. Soc. Perkins Trans. 1, p. 767 (1982)).
It would be desirable to have analogs of forskolin that would be stable and specific for forskolin binding proteins. The synthesis of such analogs could best be achieved by the use of specific intermediates that would have the following properties: 1) stability; 2) chemical groups that would be reactive so that they could be modified to produce a number of different final derivatives; and 3) reactive groups placed at positions on forskolin such that they would produce final derivatives that were specific for different forskolin binding proteins.
All patents and publications referred to herein are hereby incorporated by reference.